Cell lysis device

ABSTRACT

A micromachined cell lysis device with electrodes that are spaced by less than 10 μm from one another. The cells are attracted to the space between the electrodes and then lysed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/954,684, filed Sep. 11, 2001, which is a continuation of Ser. No.09/191,268, filed Nov. 12, 1998, which claims the benefit of the U.S.Provisional Application No. 60/065,705, filed on Nov. 14, 1997, whichare incorporated herein by reference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

The U.S. Government may have certain rights in this invention pursuantto Grant No. N66001-96-C-8632 awarded by the U.S. Navy.

BACKGROUND

It is known that an electrical field can be used to manipulate cells.Electrical manipulation of cells can be used for separating cells,holding cells, killing micro-organisms, or other operations.

Electrical manipulation of a cell is based on dielectrophoresis. Aneutral particle, such as a microbial cell, will become polarized whensubjected to a non-uniform electric field. Due to the non-uniformity ofthe field, a net force will act on the particle. This force will producemovement of the suspended cell. This phenomenon known asdielectrophoresis the inside of the cell has and holds a differentcharge than the outside of the cell.

Macro sized electroporation systems have been designed for injectinggenes into cells. See “Electroporation and Electrofusion in CellBiology,” E. Newman, A. E. Sauer, C. A. Jordan, ed. Plenum Press, NewYork, 1989. These systems often use electrical fields to make microsizedpores on cell membranes.

Cell lysis typically refers to opening a cell membrane to allow the cellinterior to come out. Cell lysing can be used to obtain intracellularmaterial for further analysis such as DNA identification.

It is known to use the science of micromachining to manipulate cells.See, for example, S. Lee, “A Study of Fabrication and Applications ofMicromachined Cell Manipulating Devices,” Ph.D. Thesis, Seoul NationalUniversity, pp. 77-81, 1996. However, no one has previously reportedusing micromachining to form a device for cell lysis. Usually, thesesystems use cuvets that have a few millimeter range electrode gap.Lysing cells with this kind of size requires a few kilovolts of voltagesource across such a gap.

Prior cell lysing has been reported using pulsed electric fields in amacrosized electroporation system. See, for example, T. Grahl and H.Markl, “Killing of Microorganisms by Pulsed Electric Fields,” Appl.Microbio. Biotechnol., 45, pp. 148-157, 1996. The disadvantages of sucha macrosized device have been described above.

J. Cheng, et al, “Preparation and Hybridization analysis of DNA/RNA fromE. Coli on Microfabriacted Bioelectronic Chips” has suggested electroniccell lysis on a chip. However, this system still required hundreds ofvolts for lysing the cell.

SUMMARY

The present disclosure describes a new micromachined cell lysis device.A microsized cell lysis device as disclosed reduces the size of theentire system including the power source, since the electrode gap couldbe reduced to a few μm or smaller. This micro-sized cell lysis device iscapable of operating on a small number of cells due to its small size.

A special way of using the electric field that can greatly simplify thepurification steps is described. This can be used to prepare biosamples.In addition, the small size allows a reduction in voltage required forlysing. The voltage can be reduced to practical levels, e.g., less than50 volts, since the electrode gap is on the order of microns.

A new structure is also described for cell lysis.

BRIEF DESCRIPTION OF THE DRAWING

These and other advantages will now be described in detail with respectto the accompanying drawings, wherein:

FIG. 1 shows a schematic view of the overall cell lysis device;

FIG. 1B shows a top view of the cell lysis electrode;

FIGS. 2A-2D show the fabrication steps of the cell lysis device;

FIG. 3 shows a photograph of a fabricated device;

FIG. 4 shows schematically the power system used for cell lysis;

FIG. 5 shows a plot of a waveform for cell lysis;

FIG. 6 shows drawings of yeast cells before and after lysing;

FIG. 7 shows a plot of lysis vs voltage.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The basic lysis device is shown in plan view in FIGS. 1 and 1B. Thedevice is made according to the fabrication steps explained below withreference to FIGS. 2A-2D.

The micromachining operates to form features on a silicon substrate.

First an insulator is formed on the silicon substrate, by oxidizing thesilicon substrate 200 to form a thermally-grown 5000 Å silicon oxidelayer 201 as shown in FIG. 2A. Chromium/gold (Cr/Au) is thermallyevaporated and patterned to form electrodes 202 on the oxidized surface.The electrodes are formed with a number of pointed portions facing oneanother, in the general shape shown in FIG. 1B.

A 4 μm thick Parylene layer is deposited and patterned to form Parylenebarriers 210 as shown in FIG. 2C. These barriers have side surfaces thathold the cell in a proper place, and form blocks between each pair ofelectrode surfaces.

FIG. 2D shows bonding the thus-made assembly to a glass substrate whichhas an inlet 220, an outlet 222, and a channel 230 between the inlet andoutlet. The channel is 30 μm high, made by timed wet etching.

The preferred device is designed for yeast cells. The distance betweenelectrodes is hence around 5 μm. More generally, the distance can rangebetween about 0.8 μm and 100 μm (0.1 mm), more preferably on the orderof e.g. 1-9.9 μm.

The final assembled device is shown in FIG. 1. A number of cells areshown, such as cell 102. Cells are attracted by the dielectrophoreticforce using an AC voltage. The cells are then lysed, using pulsedelectric fields. The AC voltage depends on the conductivity andpermitivity of the cell suspensions and the sizes of the cells. Thecells are held between two electrodes 110, 112 and between the Parylenebarriers 120, 122 for the lysing.

Any arrangement of pairs of electrodes, such as interdigitated orparallel, can be used for the cell lysing. Preferably, the edges of theelectrodes are made sharp as shown in order to concentrate the fieldbetter on the cells. The nearest distance 114 between the two electrodesis preferably equal to the mean diameter of a cell plus the standarddeviation of the cells in order to obtain the most effective lysing.

FIG. 1A shows a drawing of the electrode without the Parylene barrierspresent showing interdigitated electrodes. Distance 114 is defined asthe distance between the sharp ends of the electrodes.

FIG. 3 shows a drawing of the device from the top, showing all thearrangements of the various structures.

An important feature includes how the device is operated. A power systemfor the cell lysis is formed as shown in FIG. 4. Control is selected bya switch 400 which selects between manual mode or automatic mode. In themanual mode, the pulse is applied by a push-button switch 402. In theautomatic mode, pulses are supplied at every defined interval. Pulsewidth control is provided by a multivibrator 410, typically a TTL-typemultivibrator, part 74LS123. The switch 400 can be a single-pull,double-throw type relay.

A multipurpose function generator 420 provides the electric fields whichattracts the cells. The electric field is preferably a sinusoidal wave.A power MOSFET 422 provides the output to the cell lysis device 100.

A typical waveform is shown in FIG. 5, which shows a sample plot of thewaveform for cell lysis. The waveform includes two parts—the attractionphase 500, and the lysing phase 502.

The attraction phase uses a 6 volt AC, 2 MHZ sample. This attracts thecells to the lysing locations. A sinusoidal wave is preferably used toattract the cell to the location. After a short delay, lysing pulse, a100 μs, 20 volt pulse, is applied.

FIGS. 6A and 6B show the yeast cells before and after applying thepulsed voltage. FIG. 6A shows attraction of the yeast cells to theelectrode when the 2 MHZ 6V AC voltage in FIG. 5 is applied. FIG. 6Bshows the result of lysing. After lysing the cells, the inside andoutside of the cells are electrically connected, and they will no longerattract to the electrodes by the AC voltage.

FIG. 7 shows some representative lysing rates with different electricfields and pulse durations. The rate is increased with increased voltageand duration. Excessive pulse voltage and duration form electrolysiseffects. The optimum value for yeast cell lysing is believed to occur at100 μs and 20V. However, any voltage less than 50 volts is preferred andwithin the preferred embodiment.

Although only a few embodiments have been described in detail above,other embodiments are contemplated by the inventor and are intended tobe encompassed within the following claims. In addition, othermodifications are contemplated and are also intended to be covered. Forexample, other shapes and sizes of electrodes could be used. There couldalso be more than two electrodes. While the pointed electrodes arepreferred, flat shaped electrodes can also be used.

1. A micromachined cell lysis device, comprising: a silicon substrate;an insulator, covering at least a portion of said silicon substrate; atleast two electrodes, formed on said insulator, and between which anapplied electric field can be provided; and a distance between saidelectrodes being less than 100 μm.
 2. A device as in claim 1 whereinsaid electrodes have sharp edges, and a distance between said sharpedges is less than 100 μm.
 3. A device as in claim 2 further comprisingat least two cell blocker elements, providing physical barriers whichextend to hold a cell into place at a desired location between saidsharp edges of said two electrodes.
 4. A device as in claim 1, whereinsaid distance is less than 10 μm.
 5. A system as in claim 4 wherein adistance between sharp points of said two electrodes is substantially amean diameter of a desired cell plus a standard deviation among cells.6. A device as in claim 4 wherein a distance between electrodes is 5 μm.7. A device as in claim 3, wherein said cell blocker elements are formedof Parylene.
 8. A method of forming a cell lysis device usingmicromachining techniques, comprising: obtaining a substrate; formingtwo desired electrode patterns on the substrate, with a distance betweensaid two desired electrode patterns of less than 100 μm; and formingblocks for the cells to hold the cells at a location between saidelectrodes.
 9. A method as in claim 8, wherein said forming comprisesforming an electrode pattern including sharp edges, and wherein adistance between said sharp edges is less than 100 μm.
 10. A method asin claim 8, further comprising: applying an AC lower voltage betweensaid electrodes to attract a cell to a spot between said electrodes; andthen, after said cell is attracted, applying a spike of DC voltage, tolyse said cell.
 11. A method as in claim 10, wherein said AC voltage is6 volts AC, and said DC voltage is 20 V DC.
 12. A method as in claim 9,wherein said distance is less than 10 μm.
 13. A method as in claim 9,wherein a distance between sharp points of said two electrodes issubstantially a mean diameter of a desired cell plus a standarddeviation among cells.
 14. A method as in claim 12, wherein said cellblocker elements are formed of Parylene.
 15. A method of lysing a cell,comprising: obtaining a cell lysis device on a silicon substrate whichincludes two desired electrode patterns on the substrate, with adistance between said two desired electrode patterns of less than 100μm; and applying a first AC lower voltage between said electrodes toattract a cell to a spot between said electrodes; and then, after saidcell is attracted, applying a spike of DC higher voltage, to lyse saidcell, wherein each of said voltages is less than 50 volts.
 16. A methodas in claim 15, wherein each of said voltages is less than or equal to20 volts.